There is a need for a technology to control invasive species and pests, e.g., fish, amphibians, mollusks, crustaceans, and insects, that can replace radiation-induced sterile males for mass releases. Reliable sterilization techniques would also be valuable for the application of genetic engineering to beneficial species for improved traits (e.g., disease resistance, improved growth or development, resistance to insecticides, disruption of mechanism for disease transmission).
Traditionally, in commercial aquaculture, sterile fish have been produced by triploid induction (the addition of one extra set of chromosomes). However, triploidy is generally thought to negatively impact performance of many species, and optimal protocols are species dependent. The application of the technology is labor intensive, and it is difficult to guarantee that 100% of the fish are triploid and therefore sterile.
An easier solution would be to mediate sterility with a transgene or mutation. Several transgenic approaches to achieve sterility have been proposed and tested, but as of today, these methods have been at best partially successful. Sterility at the physiological, cellular and molecular level has not been demonstrated (Thresher et al. (2009) Aquaculture 290:104-09 and Wong & van Eenennaam (2008) Aquaculture 275:1-12).
A major obstacle is the requirement for a temporally reversing sterility to propagate the line. A solution that does not require repeated treatment at each generation would be desirable.
The majority of studies aimed at developing transgenically sterile fish have focused on methods to inactivate hormones involved in gonadal growth, differentiation and maturation. Proposed hormone targets include, the gonadotropin releasing hormone, follicule stimulating hormone, and luteinizing hormone. The silencing of these key genes should in principle lead to sexually immature and sterile fish whose fertility can be rescued by exogenous delivery of the missing hormone.
Although elegant in theory, many difficulties are inherent in this approach. A first problem is the existence of multiple hormone gene family members in some fish genomes. In addition, these hormones have biological function beyond fertility. Finally, in models that rely on knockdown technology, sterility is not 100% and reduction in fertility varies between sex and founder lines.
An alternate approach to silencing endogenous reproduction genes is to create transgenic lines with genes designed to disrupt key signaling pathways in the patterning of early embryonic development, leading to embryo death. This approach uses either gene knockdown technology, such as antisense RNA and dsRNA, or uses the misexpression of a morphogene. These embryonic disrupters are placed under the control of embryonic specific promoters, which are expressed during embryonic development.
To achieve reversibility, and allow propagation of lines, the construct is designed with a bacterial repression system placed between the promoter and the disrupter of the critical development gene. The system uses a commercially available Tet-responsive PhCMV*-11 promoter. In theory, the fish can be bred in captivity if a drug (e.g., tetracycline or a derivative thereof) is applied briefly during embryogenesis blocking the expression of the disrupter gene and providing reversible control over reproduction. To date, efforts to produce sterile lines have proven unsuccessful. Difficulties in creating these lines may be due to leakiness in the Tet responsive promoter, resulting in low levels of expression of the embryonic disrupter gene and subsequent selection against creation of founder lines. The system also requires the use of a bacterial gene and promoter system, which complicates the regulatory review process for commercialization. An additional drawback of this approach is the need to use tetracycline (or its derivatives), which will increase production cost and create environmental hazard.
The present invention addresses many of the drawbacks of earlier methods. The invention provides a transgenic technology platform capable of efficiently sterilizing different species of vertebrate and invertebrate animals. Yet the transgenic line can be easily propagated without use of potentially toxic or harmful agents. The present technology can make commercial production of beneficial species more profitable and environmentally friendly. For example, sterilization of cultured aquatic species will: 1) prevent gene flow to wild populations and colonization of new habitats by cultured non-native species (bioconfinement); 2) protect valuable lines with improved genetics; 3) increase performance by reducing the energy spent on gonad development and sexual differentiation, or by allowing production of all male populations. An all-male population can be advantageous if the males of a species grow faster than females. Sterilization methods that are essentially 100% effective will enable development of transgenic technologies with reduced risk, and promise remarkable improvements over current containment technologies.
Thus, the invention provides significant advantages, including: (i) 100% effectiveness; (ii) lower cost compared to other approaches (no labor or treatment needed to propagate or sterilize the line); (iii) the expressed gene products do not negatively impact host performance or require use of toxin genes or agents with potential health or environmental risks; (vi) broad application of the strategy among different organisms; (vii) broad range of target species using the same construct and (viii) easy propagation of transgenic lines.